Project Summary/Abstract Mitochondrial dynamics, including fission, fusion, and movement, is a fundamental mechanism in regulating mitochondrial function. Dynamin-related protein 1 (Drp1) is the major GTP hydrolyzing protein that is responsible for fission. Studies have shown that Drp1 is abundantly expressed in adult cardiac myocytes. Paradoxically, compared to numerous cell types, adult cardiac myocytes exhibit very low frequency in mitochondrial fission. This dichotomy between the abundance of Drp1 and scarcity of mitochondrial fission has prompted us to investigate the non-canonical roles of Drp1 in cardiac muscle cells. We hypothesize that Drp1 is strategically accumulated in the mitochondria associated sarcoplasmic reticulum (SR) membrane (MAM). During excitation- contraction coupling, the localized high Ca2+ in the SR-mitochondria junctions further increases the translocation of nearby cytosolic Drp1 to mitochondria. Then, Drp1 is anchored firmly in the MAM by actin. The activation of Drp1 leads to enhanced mitochondrial respiration for ATP generation as such the heart can work most effectively and sustainably. However, excessive Drp1 activation leads to persistent mitochondrial transition pore opening and excessive reactive oxygen species (ROS) generation that causes cell injury and death. The following three specific aims are proposed to test this hypothesis. Aim 1: To determine whether and how Drp1 is preferentially localized in the SR-mitochondria junctions. Aim 2: To determine the molecular mechanisms by which Drp1 regulates excitation-contraction-bioenergetics coupling. Aim 3: To determine how chronic over activation of Drp1 leads to dysfunction in the stressed heart. We will employ multiple techniques, including biochemistry (from in vitro to in situ assays), molecular biology (gene knock in or knock out, overexpression, RNA interference), cell biology (confocal, fluorescence resonance energy transfer, super-resolution microscopy, electron microscopy), cardiac physiology (echocardiogram, NMR spectroscopy), and isoproterenol infusion mouse model of cardiac hypertrophy and failure, to obtain experimental results that will lead to mechanistic insights. Successful completion of the proposed aims will allow us to introduce a new paradigm that describes the regulation of excitation-contraction-bioenergetics-ROS nexus by Drp1 through localized Ca2+ activation and actin anchoring. This fundamental signaling mechanism will describe not only how a healthy heart can perform perpetually in face of enormous workload but also why the over activation of this unique process can lead to heart failure. Finally, this new knowledge will provide new insights in developing and discovering therapeutic agents targeting Drp1-mediated signaling pathways for treating heart failure.